Regulated intramembrane proteolysis (RIP) is an ubiquitously conserved signaling mechanism in species ranging from bacteria to humans. An essential step of RIP is the site-specific cleavage of a transmembrane segment in a signaling protein by a specific membrane-embedded protease within the lipid bilayer. These intramembrane proteases are classified into four families: the serine protease rhomboid, metalloprotease Site- 2 protease (S2P), and aspartyl proteases presenilin and signal-peptide peptidase. Despite intense investigation, the structure and mechanisms of these intramembrane proteases remain largely unknown. We have initiated systematic X-ray crystallographic and biochemical analyses of the serine protease rhomboid and the metalloprotease S2P. Significant preliminary results have been achieved;the work proposed here will build on our preliminary results with the following specific aims. (1) Determination of the crystal structure of the transmembrane core domain of the E. coli rhomboid protease GlpG in complex with inhibitor and substrate. (2) Determination of the crystal structure of full-length rhomboid proteases from prokaryotic and eukaryotic species. (3) Determination of the crystal structure of the transmembrane core domain of a S2P intramembrane protease from M. jannaschii. (4) Determination of the crystal structure of the full-length S2P intramembrane protease from M. jannaschii and from E. coli. (5) Determination of the crystal structure of a S2P intramembrane protease in complex with a substrate peptide. These studies, when completed, will reveal significant insights into the structure, function, and mechanism of the rhomboid and S2P families of intramembrane proteases. PUBLIC HEALTH RELEVANCE: Regulated intramembrane proteolysis (RIP) is an ubiquitously conserved signaling mechanism in organisms ranging from bacteria to humans. An essential step of RIP is the site-specific cleavage of a transmembrane segment in a signaling protein by a specific membrane-embedded protease within the lipid bilayer. This proposal seeks to understand the structures and mechanisms of these intramembrane proteases.